Identification of Naegleria fowleri in domestic water sources by nested PCR

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.     

Elevated interstitial adenosine concentrations do not activate the muscle reflex

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 +/- 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 microM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated (P < 0.05) from 0.9 +/- 0.3 microM during control blood perfusion to 2,421 +/- 547 microM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased (P < 0.05) from 1.1 +/- 0.3 microM during control blood perfusion to 4.1 +/- 1.2 microM during perfusion with 100 microM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings (J Appl Physiol 83: 1045-1053, 1997).     

Effects of dynamic exercise on mean blood velocity and muscle interstitial metabolite responses in humans

We examined the effects of dynamic one-legged knee extension exercise on mean blood velocity (MBV) and muscle interstitial metabolite concentrations in healthy young subjects (n = 7). Femoral MBV (Doppler), mean arterial pressure (MAP) and muscle interstitial metabolite (adenosine, lactate, phosphate, K(+), pH, and H(+); by microdialysis) concentrations were measured during 5 min of exercise at 30 and 60% of maximal work capacity (W(max)). MAP increased (P < 0.05) to a similar extent during the two exercise bouts, whereas the increase in MBV was greater (P < 0.05) during exercise at 60% (77.00 +/- 6.77 cm/s) compared with 30% W(max) (43.71 +/- 3.71 cm/s). The increase in interstitial adenosine from rest to exercise was greater (P < 0.05) during the 60% (0.80 +/- 0.10 microM) compared with the 30% W(max) bout (0.57 +/- 0.10 microM). During exercise at 60% W(max), interstitial K(+) rose at a greater rate than during exercise at 30% W(max) (P < 0.05). However, pH increased (H(+) decreased) at similar rates for the two exercise intensities. During exercise, interstitial lactate and phosphate increased (P < 0.05) with no difference observed between the two intensities. After 5 min of recovery, MBV decreased to baseline levels after exercise at 30% W(max) (4.12 +/- 1.10 cm/s), whereas MBV remained above baseline levels after exercise at 60% W(max) (Delta19.46 +/- 2.61 cm/s; P < 0.05). MAP and interstitial adenosine, K(+), pH, and H(+) returned toward baseline levels. However, interstitial lactate and phosphate continued to increase during the recovery period. Thus an increase in exercise intensity resulted in concomitant changes in MBV and muscle interstitial adenosine and K(+), whereas similar changes were not observed for MAP or muscle interstitial pH, lactate, or phosphate. These data suggest that K(+) and/or adenosine may play an active role in the regulation of skeletal muscle blood flow during exercise.     

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.     

Interstitial pH, K(+), lactate, and phosphate determined with MSNA during exercise in humans

The purpose of the present study was to use the microdialysis technique to simultaneously measure the interstitial concentrations of several putative stimulators of the exercise pressor reflex during 5 min of intermittent static quadriceps exercise in humans (n = 7). Exercise resulted in approximately a threefold (P < 0.05) increase in muscle sympathetic nerve activity (MSNA) and 13 +/- 3 beats/min (P < 0.05) and 20 +/- 2 mmHg (P < 0.05) increases in heart rate and blood pressure, respectively. During recovery, all reflex responses quickly returned to baseline. Interstitial lactate levels were increased (P < 0.05) from rest (1.1 +/- 0.1 mM) to exercise (1. 6 +/- 0.2 mM) and were further increased (P < 0.05) during recovery (2.0 +/- 0.2 mM). Dialysate phosphate concentrations were 0.55 +/- 0. 04, 0.71 +/- 0.05, and 0.48 +/- 0.03 mM during rest, exercise, and recovery, respectively, and were significantly elevated during exercise. At the onset of exercise, dialysate K(+) levels rose rapidly above resting values (4.2 +/- 0.1 meq/l) and continued to increase during the exercise bout. After 5 min of contractions, dialysate K(+) levels had peaked with an increase (P < 0.05) of 0.6 +/- 0.1 meq/l and subsequently decreased during recovery, not being different from rest after 3 min. In contrast, H(+) concentrations rapidly decreased (P < 0.05) from resting levels (69.4 +/- 3.7 nM) during quadriceps exercise and continued to decrease with a mean decline (P < 0.05) of 16.7 +/- 3.8 nM being achieved after 5 min. During recovery, H(+) concentrations rapidly increased and were not significantly different from baseline after 1 min. This study represents the first time that skeletal muscle interstitial pH, K(+), lactate, and phosphate have been measured in conjunction with MSNA, heart rate, and blood pressure during intermittent static quadriceps exercise in humans. These data suggest that interstitial K(+) and phosphate, but not lactate and H(+), may contribute to the stimulation of the exercise pressor reflex.     

Bilirubin as an antioxidant in micelles and lipid bilayers: its contribution to the total antioxidant capacity of human blood plasma

The antioxidant capacities, antioxidant activities, k(inh), and stoichiometric factors, n, of water-soluble derivatives of bilirubin (BR), BR-human serum albumin (BR-HSA), and BR-ditaurate disodium conjugate (BRC) were determined in aqueous/lipid dispersions of sodium dodecyl sulfate (SDS) micelles/methyl linoleate and in bilayers of dilinoleoylphosphatidylcholine (DLPC) during initiation by water-soluble azo-bis-amidinopropane dihydrochloride (ABAP). The inhibition rate constants for BRC and BR-HSA were similar in micelles (k(inh) approximately 1.3 x 10(4) M(-1) s(-1)), where n approximately 2, whereas the k(inh) for BR-HSA dropped by (1/2) in bilayers. The dimethyl ester of bilirubin (BRDE) gave a k(inh) only one-tenth that of the vitamin E analog, pentamethylhydroxychroman (PMHC) in SDS micelles/methyl linoleate when initiated by lipid-soluble azo-bis-2,4-dimethylvaleronitrile (DMVN). Biliverdin hydrochloride (BVHCl) was NOT an effective peroxyl radical-trapping agent in the micellar phase during initiation by ABAP or DMVN containing methyl linoleate but it inhibited oxygen uptake in the aqueous phase. Both BRC and BR-HSA extended the total radical antioxidant parameter (TRAP) of human blood plasma and their contribution to TRAP was in the range of 5-10% of the natural TRAP of blood plasma, depending on the BR content determined in the blood plasma.